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Design and Engineering Next-Generation Nanopore Devices for Bioplymer Analysis

Periodic Reporting for period 2 - DeE-Nano (Design and Engineering Next-Generation Nanopore Devices for Bioplymer Analysis)

Reporting period: 2019-01-01 to 2020-06-30

The main objective of this work is to design and engineer nanopores that will be used in biopolymer analysis. We decided to focus on polypeptides, with the ultimate goal of sequencing single proteins.
The ability of sequencing proteins is important in practical and base science. Proteins are biomarkers linked to disease, hence devices capable of identify proteins are important in medical diagnostics. In addition, while all cells have the same DNA, the expression of proteins is unique. Hence, if we want to understand how a cell works, we need to understand how proteins are made and modified. To date there is no single-molecule technique to sequence proteins. Single-molecule sequencing is important because proteins exist in cells in highly variable and heterogeneous mixtures. And only single-molecule techniques can address this issues.
The overall objective of this proposal is to understand how to design and engineer nanopores ta can process biopolymers. The preparation of nanopores is fundamental if we want to sequence proteins. Indeed, one of the main limitations in the field is that very few nanopores exist in nature that can be used. Hence we are addressing here of the problem of making these nanopores. We first start with designing de novo proteins that can insert into lipid bilayers, to then move towards more complex system that also will include a nanomachine that is capable of unfloding and threading polypeptides.
In WP1 we aimed at understanding the rules of introducing a protein inside a lipid bilayer. Thus far, we were ca[able of inserting an artificial transmembrane beta barrel in two separate proteins. Hence we have completed WP1.
In the second WP we aimed at creating an artifical nanopore-unfoldase. We also have prepared one of constructs. Others are in preparation. We are at the moment validating these constructs.
WP 3 was dedicated to the recognition of amino acids. We showed that post-translational modifications can be identified by nanopore currents. We also showed that single amino acid differences can be observed by nanopore currents. Much work is still dedicated in the engineering of nanopores for improving the identification of polypeptides
We have described how to design a new nanopore. This was never described before in the literature. In particular we have identified a reliable method to add a beta barrel into a solubile protein and inserte into a lipid bilayer.
We also have shown that nanopore currents can be used to identify the mass of peptides. This is an unexpected and important discovery, as it open to the making nanopores mass spectrometry devices.